Resultado final Production and characterization of ET proteins The modification, production and characterization of variants of a series of blue copper proteins and redox enzymes was undertaken. Cu proteins were produced in the native form, the apo-form, and the Zn-containing form. The proteins were tailored to user specifications as the project developed. New variants could be offered as functional characterisation of the protein and enzyme variants progressed in Leiden. In addition clones of protein variants were provided on demand. The mutations applied to the various proteins and enzymes pertained to the engineering of surface cysteines, allowing one-, two- or three-point immobilizations at solid surfaces, and the creation of cavity mutants allowing the use of hot wires. Additional work related to the synthesis of hot wires and the construction of specialised clones. The design of new variants was routinely based on extensive modeling. Construction at the DNA-level was achieved by state-of the-art rec-DNA techniques. Expression and purification of the proteins/enzymes was achieved according to advanced protein chemistry techniques. Purification protocols were developed or adjusted where necessary. Purification protocols for the Cys-containing variants were developed with a special eye on keeping the engineered cysteine(s) active for immobilization on solid substrates. Characterisation of the proteins/enzymes was achieved by standard biochemical techniques. Spectroscopic characterisation involved UV-VIS spectroscopy, mass spec analysis, protein film voltammetry, cyclic voltammetry and, when necessary, EPR and NMR spectroscopy. Activity was checked by enzymological and kinetic techniques (stopped flow, NMR). At a later stage of the project proteins/enzymes were also characterised for their behaviour on solid supports (gold, mica, carbon) by means of atomic force microscopy (AFM) both under ambient conditions and in situ. Variants were prepared of azurin from Pseudomonas aeruginosa, of pseudo-azurin from Alcaligenes faecalis S-6, of plastocyanin from Populus nigra and of amicyanin from P. versutus. The enzymes Nitrite reductase (NiR) from Alcaligenes faecalis S-6 and Methylamine dehydrogenase from Paracoccus versutus were produced and NiR-variants were constructed and produced that contained cysteines at various positions. In addition Heme-cd1-containing NiR and Nitric oxide reductase were made available. Details are provided in the overview below. Overview of Metallo-proteins, wt and variants produced for SAMBA: ET Metallo-proteins: wild types & variants Azurin [Az, small cupredoxin]: wild type forms & variants -- Wild type forms (Cu, Zn, apo) -- Single surface cysteine: Q12C, K27C (Cu, Zn, apo), N42C, S118C -- Click-on chemistry cavity mutant H117G -- Removal S-S bridges: C3AC26A Plastocyanin[Pc, small cupredoxin]: wild type forms & variants -- Engineered S-S bridge: I21C, E25C (PCSS) -- C-terminal extension TCG (Thr-Cys-Gly) (PCSH) Pseudoazurin [PsAzu, small cupredoxin]: wild type forms & variants -- Single & Double surface cysteines: E51C, E54C, -- E51CE54C Amicyanin [Ami, small cupredoxin]: wild type forms Enzymes: wild types & variants Copper-containing nitrite reductase [NiR, multicopper oxidase]: wild type forms & variants: -- Type-1 site depleted, Type-2 site depleted -- Single surface cysteine: L93C, M94C -- Click-on chemistry cavity H145A/G, M150G, H306A Heme/cd1 containing nitrite reductase [cd1NiR, multi heme enzyme] :wild type Nitric oxide reductase [Nor, multi heme enzyme] :wild type Methylamine dehydrogenase [MADH ] : wild type Others: Azurin antibodies Hotwire linker (1-(11-mercapto-undecyl)-imidazole dimer) Plasmid containing the gene of azurin. Demonstration of electronic devices based on metalloproteins working in air and at room temperature We demonstrated the possibility of implementing different types of biomolecular electronic devices based on metalloproteins with functionality like rectification and amplification of electrical signals. The results may contribute to build future alternative nanoelectronic devices. The devices are based on silicon /silicon dioxide substrates onto which small metallic nanoelectrodes are defined by means of advanced lithographic processes and nanofabrication methods. The proteins are chemisorbed onto the substrates. To reach this goal a strong and proficient collaboration among the project parteners has been set up in order to produce the molecules, to understand the self-assembly of the molecular layer and the chemisorption process and to investigate protein stability. Strong enphasis was given to the set up of new protocols for the fabrication and testing of nanodevices. Theoretical calculations and models of the transport mechanisms in protein devices were also carried out. Biosensors: The adsorption of genetically-modified azurin on single crystal faces of gold was examined by AFM, STM and electrochemistry The adsorption of genetically-modified azurin on single crystal faces of gold was examined by AFM, STM and electrochemistry (Davis et al., Chem. Commun., 2003, 576-577). This work was extended to include another copper protein, plastocyanin. Images of individual molecules were observed and the rate of electron transfer measured (Andolfi et al., J. Electroanal. Chem., 2004, 565, 21-28). A method of using gold electrodes, surface-modified with cysteine-containing peptides allowed electrochemical investigations of pseudoazurin and permitted a brief examination of its interaction with a nitrite reductase, (Astier et al., Electroanalysis, 2004, 16, 1155-1165). This was extended (Astier et al., ChemPhysChem., 2005, 6, 1114-1120) showing that this system provides a method of analysis for the nitrite anion in the micromolar range. This work demonstrated that one could gain an intimate knowledge of the mode of adsorption of a key electron transfer protein and then use this knowledge to fashion a sensor, in this case for nitrite, employing an enzyme for which the latter was a substrate. Of course, in order to translate this into a commercial sensor, there are many developments that need to occur, e.g., regarding the nature of the electrode, the stability of the proteins etc. However, in principle, an enzyme electrode in which the sole working entities are biomaterials has been demonstrated. To be capable of development, an industrial partner is necessary: the device chosen may not be a nitrite sensor but the lessons are transferable. It has been particularly valuable to interact with our other partners, particularly Professors Canters and Cannistrato. Buscando datos de OpenAIRE... Se ha producido un error en la búsqueda de datos de OpenAIRE No hay resultados disponibles
